Method and device for electrochemical immunoassay of multiple analytes

ABSTRACT

A method and device for detection and quantification of biologically significant analytes in a liquid sample is described. The method includes contacting a volume of a liquid sample with predetermined amounts of at least a first and second redox reversible species having redox potentials differing by at least 50 millivolts. At least one of the redox reversible species comprises a liquid sample diffusible conjugate of a ligand analog of an analyte in the liquid sample and a redox reversible label. A predetermined amount of at least one specific binding partner for each analyte to be measured is combined with the sample and current flow is measured at first and second anodic and cathodic potentials and correlated with current flows for known concentrations of the respective diffusible redox reversible species. Diagnostic devices and kits, including such devices and the specified specific binding partner(s) and redox reversible species are also described.

This application claims benefit of provisional application 60/087,576Jun. 1, 1998.

FIELD OF THE INVENTION

This invention relates to a method and device for detection andquantification of biologically significant analytes in a liquid sample.More particularly the invention is directed to a biosensor and method ofusing same for electrochemical immunoassays of multiple analyte speciesin a single liquid sample.

BACKGROUND AND SUMMARY OF THE INVENTION

Therapeutic protocols used today by medical practitioners in treatmentof their patient population requires accurate and convenient methods ofmonitoring patient disease states. Much effort has been directed toresearch and development of methods for measuring the presence and/orconcentration of biologically significant substances indicative of aclinical condition or disease state, particularly in body fluids such asblood, urine or saliva. Such methods have been developed to detect theexistence or severity of a wide variety of disease states such asdiabetes, metabolic disorders, hormonal disorders, and for monitoringthe presence and/or concentration of ethical or illegal drugs. Morerecently there have been significant advancements in the use ofaffinity-based electrochemical detection/measurement techniques whichrely, at least in part, on the formation of a complex between thechemical species being assayed (the “analyte”) and another species towhich it will bind specifically (a “specific binding partner”). Suchmethods typically employ a labeled ligand analog of the target analyte,the ligand analog selected so that it binds competitively with theanalyte to the specific binding partner. The ligand analog is labeled sothat the extent of binding of the labeled ligand analog with thespecific binding partner can be measured and correlated with thepresence and/or concentration of the target analyte in the biologicalsample.

Numerous labels have been employed in such affinity based sampleanalysis techniques, including enzyme labeling, radioisotopic labeling,fluorescent labeling, and labeling with chemical species subject toelectrochemical oxidation and/or reduction. The use of redox reversiblespecies, sometimes referred to as electron transfer agents or electronmediators as labels for ligand analogs, have proven to provide apractical and dependable results in affinity-based electrochemicalassays. However, the use of electrochemical techniques in detecting andquantifying concentrations of such redox reversible species (correlatingwith analyte concentrations) is not without problem. Electrochemicalmeasurements are subject to many influences that affect the accuracy ofthe measurements, including those relating to variations in theelectrode structure itself and/or matrix effects deriving fromvariability in liquid samples.

The present invention relates to immunosensors based on directelectrochemical measurement of detectable species with microarrayelectrodes under bipotentiostatic control. An electrochemical label, forexample an Os mediator, is covalently attached to a peptide which hasamino acid sequence of the binding epitope for the antibody. Whenindicator/peptide conjugate is bound to antibody, the indicator does notfunction electrochemically or it is said to be “inhibited”. The analytepresent in sample will compete with indicator/peptide conjugate for thelimited number of binding sites on the antibody. When more analyte ispresent, more free indicator/peptide conjugate will be left producinghigher current at a sensor electrode, i.e., one of the workingelectrodes where measured events (oxidation or reduction) are takingplace. In the opposite case, when less analyte is present, moreindicator/peptide conjugate will be bound to antibody resulting lessfree conjugates and producing lower current levels at the workingelectrodes. Therefore the current detected at either one of the workingelectrodes will be a function of analyte concentration.

It is frequently desired to measure more than one analyte species in aliquid sample. Measurement of multiple species in a mixture has beenachieved with photometry and fluorescence, via selection of theappropriate wavelengths. Electrochemical measurements of a singlespecies in a complex mixture are routinely made by selecting a potentialat which only the desire species is oxidized or reduced (amperometry) orby stepping or varying the potential over a range in which only thedesired species changes its electrochemical properties (AC and pulsemethods). These methods suffer from disadvantages including lack ofsensitivity and lack of specificity, interference by charging and matrixpolarization currents (pulse methods) and electrode fouling due to theinability to apply an adequate overpotential. Moreover, electrochemicalmeasurements are complicated by interference between the multiplicity ofelectroactive species commonly extant in biological samples.

Electrode structures which generate steady state current via diffusionalfeedback, including interdigitated array electrodes (IDAs) (FIGS. 1 and2) and parallel plate arrangements with bipotentiostatic control areknown. They have been used to measure reversible species based on thesteady state current achieved by cycling of the reversible species. Areversible mediator (redox reversible species) is alternately oxidizedand reduced on the interdigitated electrode fingers. The steady statecurrent is proportionate to mediator concentration (FIG. 3) and limitedby mediator diffusion. A steady state current is achieved within secondsof applying the predetermined anodic (more positive) and cathodic (lesspositive or negative) potentials (FIG. 6) to the microelectrode array.The slope of a plot of the IDA current vs. mediator concentration isdependent on IDA dimensions, and the slope increases with narrowerelectrode spacings (FIG. 7).

One embodiment of the present invention provides a method for measuringmultiple analyte species in the same sample, and optimally on the sameelectrode structure, thus improving the accuracy of the relativemeasurements. This invention also provides an electrochemical biosensorwith capacity to provide improved accuracy through the use ofself-compensation. Analyte concentration can be measured/calculated fromelectrometric data obtained on the same liquid sample with the sameelectrode structure (the working electrodes), thereby minimizingperturbations due to variability in sample or electrode structure.

The various embodiments of this invention utilize the principle ofdiffusional recycling, where a diffusible redox reversible species isalternately oxidized and reduced at nearby electrodes, therebygenerating a measurable current. As alternate oxidation and reduction isrequired for measurement, only electroactive species which areelectrochemically reversible are measured thereby eliminating, or atleast reducing, the impact or interference from non-reversibleelectroactive species in the sample. Redox reversible species havingdifferent oxidation potentials can be independently measured in amixture by selecting and bipotentiostatically controlling the oxidizingand reducing potentials for neighboring electrode pairs so that only thespecies of interest is oxidized at the anode (the electrode with themore positive potential) and reduced at the cathode (the electrode withthe less positive or negative potential). When the working electrodes(the anode/cathode arrays) are dimensioned to allow diffusionalrecycling of the redox-reversible-species at the selected oxidizing andreducing potentials appropriate for that species, a steady state currentat the working electrodes where the measurable oxidative and reductiveevents are taking place, is quickly established through the sample andthe electrode structure. The magnitude of the current is proportional tothe concentration of the diffusible redox reversible species in thesample. When two or more redox reversible species are utilized, they areselected to have redox potentials differing by at least 50 millivolts,most preferably at least 200 millivolts, to minimize interferencebetween one species and the other in measurements of the respectivesteady state currents.

Any electrode structure which allows for diffusional recycling toachieve steady state current in response to application of pre-selectedspecies-specific anodic and cathodic potentials can be utilized incarrying out the invention. Suitable electrode structures includeinterdigitated array microelectrodes and parallel plate electrodesseparated by distances within the diffusion distance of the respectiveredox reversible species. The electrode structures typically include areference electrode (e.g., Ag/AgCl), at least two working electrodes(one at positive potential and another at a less positive or negativepotential relative to the reference electrode), and optionally anauxiliary electrode for current control. In use, a programmablebipotentiostat is placed in electrical communication with the electrodestructure for applying the respective anodic and cathodic potentialsspecific for each of the respective redox reversible species utilized inthe method/biosensor. Several novel osmium complexes have been developedfor use as labels for preparing ligand analog conjugates havingpotential differences sufficient to allow the use of two osmiumcomplexes (as opposed to an osmium complex and a ferrocene or otherredox reversible label) in this invention.

Accordingly, one embodiment of the invention provides a device fordetecting or quantifying one or more analytes in a liquid sample. Thedevice comprises at least two redox reversible species having respectiveredox potentials differing by at least 50 millivolts, and an electrodestructure for contact with the liquid sample. In one embodiment thedevice further comprises a chamber for containing the liquid sample,optionally dimensioned for capillary fill. The electrode structureincludes a reference electrode and an anode and a cathode (workingelectrodes) dimensioned to allow diffusional recycling of the redoxreversible species in the sample when aredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a redox-reversible-species-dependent anodicpotential is applied to a second electrode to enable and sustain ameasurable current through the sample. The device also includesconductors communicating with the respective electrodes for applyingpotentials and for carrying current conducted between the sample and therespective electrodes.

The device in accordance with this invention is typically utilized incombination with a meter which includes a power source, for example abattery, a microprocessor, a register for storing measured currentvalues, and a display for reporting calculated analyte concentrationsbased on measured current values. The construction and configuration ofsuch meters are well known in the art. Meters for use in accordance withthe present device further comprise a bipotentiostat under control ofthe microprocessor or separately programmable to apply predeterminedpotentials to the device component microelectrode arrays during liquidsample analysis. Improvements in meter construction and design forbiosensor systems are described in U.S. Pat. Nos. 4,999,632; 5,243,516;5,366,609; 5,352,351; 5,405,511; and 5,48,271, the disclosures of whichare hereby incorporated by reference.

In another embodiment of the invention there is provided a method formeasuring the concentration of one or more analytes in a liquid sample.The method includes contacting a portion of the sample withpre-determined amounts of at least a first and second redox reversiblespecies having a redox potential differing by at least 50 millivoltsfrom that of each other species. Each respective species comprises aliquid sample diffusible conjugate of a ligand analog of an analyte inthe liquid sample and a redox reversible label. The liquid sample isalso contacted with a predetermined amount of at least one specificbinding partner for each analyte to be measured. The diffusibleconjugate is selected so that it is capable of competitive binding withthe specific binding partner for said analyte.

The concentration of diffusible redox-reversible-species in the liquidsample is then determined electrochemically. The sample is contactedwith an electrode structure, including a reference electrode and atleast first and second working electrodes dimensioned to allowdiffusional recycling of at least one of the diffusibleredox-reversible-species in the sample, when a predeterminedredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to the second working electrode. Typically,a first cathodic potential is applied to the first working electrode anda first anodic potential is applied to the second working electrode toestablish current flow through the sample due to diffusional recyclingof the first redox-reversible-species without significant interferencefrom the second redox-reversible-species. Current flow through one ormore of the electrodes at the first anodic and cathodic potentials ismeasured. Similarly current flow responsive to application of secondcathodic and anodic potentials to electrodes in contact with the sampleis measured and correlated with measured current flows for knownconcentrations of the respective redox-reversible-species, saidconcentrations being proportionate to the respective analyteconcentrations at a predetermined redox-reversible species-dependentpotential (anodic or cathodic). Alternatively, the potential of one ofthe working electrodes can be held constant and current flow ismonitored as the potential of the other working electrode is varied andswept through the other redox-reversible species-dependent potential.

The reagent components for the invention, including the redox reversiblespecies and the specific binding partners, can be provided in the formof a test kit for measuring the targeted analyte(s) in a liquid sample,either as separate reagents or, more preferably, combined as amulti-reagent composition, e.g. combined redox reversible species,combined specific binding partners, or combined redox reversible speciesand specific binding partners. The kit optionally, but preferably,includes an electrode structure dimensioned to allow diffusional redoxrecycling of diffusible redox reversible species in the liquid sample.The electrode structure includes conductors for connecting the structurebipotentiostat programmed to applyredox-reversible-species-dependent-anodic and cathodic potentials to theelectrode structure and to sense and measure current flow, typically atone or both of the working electrodes, responsive to such appliedpotentials.

Also described herein is the preparation and use of electrochemicallydetectable osmium complexes and covalent conjugates of said complexeshaving oxidation potentials differing sufficiently to enable their usetogether in the respective method and device embodiments of theinvention. Osmium labeled ligand analogs capable of binding to aspecific binding partner of a biologically significant analyte areprepared. One group of electrochemically detectable conjugates comprisea bis(bipyridyl) imidazolyl chloroosmonium complex characterized by fastmediation kinetics and low redox potential (+15 mV vs. Ag/AgCl). Anothergroup of osmium complex labeled, electrochemically detectable conjugatesinclude tris(bipyridyl) osmium complexes, which, like the bis(bipyridyl)imidazolyl chloroosmium complexes are characterized by fast mediationkinetics, but the tris(bipyridyl) complexes have a redox potentialsufficiently different from the bis(pyridyl) imidazolyl chloroosmiumcomplexes to allow their use together in the various embodiments of thisinvention to enable use of microelectrode arrays for measuring more thanone analyte in a single liquid sample by concentration dependentcurrents amplified by diffusional redox recycling.

In one preferred embodiment of the invention at least one osmium complexconjugates is used in combination with another conjugatedredox-reversible-species for the measurement of both glycosylatedhemoglobin and hemoglobin in a lysed blood sample. Oneredox-reversible-species preferably comprises an osmium complexcovalently linked to a ligand analog of either hemoglobin orglycosylated hemoglobin, and the second redox-reversible-speciescomprises a second redox reversible label covalently bound to a ligandanalog of the other of the two target analytes. The method enablesmeasurement of the concentration of both the glycosylated hemoglobin(HbAlc) and the concentration of either total hemoglobin or that ofunglycosylated hemoglobin (HbA₀) thereby enabling calculation of theresults as a ratio of the two measurements (% HbAlc). It is advantageousto assay both HbAlc and total hemoglobin (or HbA₀) using the sameprinciple in a single sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an enlarged plan view of an interdigitated array electrode forreversible mediator measurement in accordance with the presentinvention.

FIG. 2 is a partial cross-sectional view of the electrode of FIG. 1illustrating the conditions of steady state current limited by diffusionof reversible mediator (M) which is alternately oxidized and reduced onthe interdigitated electrode fingers.

FIG. 3 is a graphic presentation of dose response currents for abis-(bipyridyl) imidazolyl chloroosmonium mediator in a peptideconjugate of that mediator.

FIG. 4 is a graphic illustration of current flow vs. concentration ofglycosylated hemoglobin (HbAlc) in blood samples using an osmiumconjugate and enzyme amplified DC amperometry.

FIG. 5 is a graphic illustration of the inhibition of current flow dueto free conjugate as a function of antibody concentration (C_(n)) asmeasured using enzyme amplified DC amperometry [C₁>C₂>C₃].

FIG. 6 is a graphic illustration of current flow vs. time using aninterdigitated array electrode.

FIG. 7 is a graphic illustration of the effect of the dimensions of theinterdigitated array electrode structure on current flow as a functionof concentration of an osmium conjugate (Os-DSG-Alc).

FIG. 8 is a graphic illustration of current flow as a function ofapplied potential for a liquid sample containing equimolar (50 μM) of abis-(bipyridyl) imidazolyl chloroosmium complex and a tris(bipyridyl)osmium complex.

FIG. 9 is a graphic presentation of current flow vs. concentration of aferrocene-biotin conjugate in the presence of varying amounts of anosmium complex conjugate on interdigitated array electrodes withbipotentiostatic control.

FIG. 10 is a graphic illustration of the effect of concentration of anunlabeled conjugate (BSA-Alc) on current flow in a solution containingosmium labeled conjugate (osmium-DSG-Alc)) in the presence of threeseparate Alc-recognizing antibody compositions.

FIG. 11 illustrates the structure of a tris(bipyridyl) osmium labeledconjugate for use in accordance with this invention.

FIGS. 12-14 are similar and each depict the chemical structure of abis(bipyridyl) imidazolyl chloroosmium labeled peptide conjugate for usein accordance with this invention.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention is a method for measuring the concentrationof one or more analytes in a liquid sample. The method enables two ormore independent amperometric measurements of the sample on a singleelectrode structure.

The method comprises

contacting a volume of said liquid sample with

1) predetermined amounts of at least a first and second redox reversiblespecies, each respective species having a redox potential differing byat least 50 millivolts from that of each other species, at least onespecies comprising a liquid sample diffusible conjugate of a ligandanalog of an analyte in the liquid sample and a redox reversible label,said conjugate capable of competitive binding with a specific bindingpartner for said analyte, and

2) a predetermined amount of at least one specific binding partner foreach analyte to be measured; and

electrochemically determining the concentration of each of saiddiffusible redox-reversible species in the liquid sample by

contacting said sample with an electrode structure including a referenceelectrode and at least first and second working electrodes dimensionedto allow diffusional recycling of the diffusible redox reversiblespecies in the sample when a predetermineredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to a second working electrode, saiddiffusional recycling of said species being sufficient to sustain ameasurable current through said sample, applying a first cathodicpotential to the first working electrode and a first anodic potential tothe second working electrode, said first cathodic and anodic potentialscorresponding to those respective potentials necessary to establishcurrent flow through the sample due to diffusional recycling of thefirst redox reversible species without significant interference fromsaid second redox reversible species,

measuring current flow at said first anodic and cathodic potentials,

applying a second cathodic potential to said first or second workingelectrode and a second anodic potential to the other working electrode,said second cathodic and anodic potential corresponding to thoserespective potentials necessary to establish current flow through thesample due to diffusional recycling of the secondredox-reversible-species without significant interference from the firstredox reversible species,

measuring current flow at said second anodic and cathodic potentials,and

correlating the respective measured current flows to that for knownconcentrations of the respective diffusible redox reversible species.

The method of the invention has very broad applicability but inparticular may be used to assay: drugs, hormones, including peptidehormones (e.g., thyroid stimulating hormone (TSH), luteinizing hormone(LH), follicle stimulating hormone (FSH), insulin and prolactin) ornon-peptide hormones (e.g., steroid hormones such as cortisol,estradiol, progesterone and testosterone, or thyroid hormones such asthyroxine (T4) and triiodothyronine), proteins (e.g., human chorionicgonadotropin (hCG), carcino-embryonic antigen (CEA) and alphafetoprotein(AFP)), drugs (e.g., digoxin), sugars, toxins or vitamins.

The method can be performed on liquid samples comprising biologicalfluids such as saliva, urine, or blood, or the liquid sample can bederived from environmental sources. The liquid samples can be analyzed“as is,” or they can be diluted, buffered or otherwise processed tooptimize detection of the targeted analyte(s). Thus, for example, bloodsamples can be lysed and/or otherwise denatured to solubilize cellularcomponents.

The method can be performed using widely variant sampling handlingtechniques. Thus, the sample can be premixed with either or both of thespecific binding partner for the targeted analytes and the redoxreversible species prior to contacting the sample with the electrodestructure, or the liquid sample, either neat or pre-processed, can bedelivered to a vessel containing predetermined amounts of the redoxreversible species and the specific binding partner for subsequent orsimultaneous contact with the electrode structure. The order ofintroduction of the components into the sample is not critical; however,in one embodiment of the invention the predetermined amounts of thespecific binding partners are first added to the sample, and thereafter,there is added the predetermined amounts of the redox reversiblespecies. It is also possible to combine the predetermined amounts of thespecific binding partners with the redox reversible species to form therespective complexes prior to combining those components with the liquidsample. In that latter case the redox reversible species will bedisplaced from its respective specific binding partner by thecorresponding analyte to provide a concentration of the redox reversiblespecies proportionate to the concentration of analyte in the liquidsample. The reagents, that is, the predetermined amounts of the specificbinding partner of each analyte and the predetermined amounts of thecorresponding redox reversible species can, for example, be deposited ina vessel for receiving a predetermined volume of the liquid sample. Theliquid sample is added to the vessel, and thereafter, or simultaneously,the liquid sample is contacted with the electrode structure.

The electrode structure includes a reference electrode and at leastfirst and second working electrodes dimensioned to allow diffusionalrecycling of the diffusible redox reversible species in the sample whenpredetermined redox-reversible-species-dependent-cathodic and anodicpotential is applied to the working electrodes. The term “workingelectrode” as used herein refers to an electrode where measured events(i.e. oxidation and/or reduction) take place and resultant current flowcan be measured as an indicator of analyte concentration. “Anodicpotential” refers to the more positive potential (applied to the anode)and “cathodic potential” refers to the less positive or negativepotential applied to the cathode (vs. a reference electrode). Electrodesdimensioned to allow diffusional recycling are well known in the art andare typically in the form of arrays of microdiscs, microholes, ormicrobands. In one embodiment the electrodes are in the form of aninterdigitated arrangement of microband electrodes with micron orsubmicron spacing. Short average diffusional length and a large numberof electrodes are desirable for effective current amplication byrecycling of reversible redox species. The microelectrode arrays can befabricated, for example, as pairs of interdigitated thin film metalelectrodes in micron and submicron geometry arranged on an insulatorsubstrate, for example, oxidized silicon. Each of the electrode fingers(FIG. 1) are spaced from its neighboring finger in the nanometer to lowmicrometer (1-10 microns) range. Microelectrode arrays can be fabricatedusing photolithography, electron bean lithography, and so-calledlift-off technique. Thus, an interdigitated electrode array (IDA) can bedeposited on glass, silicon or polyamide utilizing the following generalprocedure:

1. Grow thermal oxide layer on silicon substrate;

2. Sputter 400 Å chromium seed layer, 2000 Ågold;

3. Spin-coat and soft-bake photo resist;

4. Expose and develop photo resist with IDA pattern;

5. Pattern gold and chromium with ion beam milling;

6. Strip photo resist; and

7. Cut electrodes into chips by first coating with a protective layer,cutting into strips, stripping the protective layer, and cleaningelectrode surfaces in oxygen plasma.

The electrode structure can be formed on an inner surface of a chamberfor receiving the liquid sample, e.g., a cuvette, a capillary fillchamber, or other sample receiving vessel wherein the electrodestructure can be contacted with the liquid sample. Alternatively, theelectrode structure can form part of a probe for dipping into the liquidsample after the sample has been contacted with the predeterminedamounts of the redox reversible species and the specific bindingpartners. The electrode structure is in contact with conductors thatenable application of the respective cathodic and anodic potentials forcarrying out the present method. The anodic and cathodic potentials areapplied relative to a reference electrode component of the electrodestructure using a bipotentiostat. The electrode structure can optionallyinclude an auxiliary electrode for current control. The bipotentiostatis utilized to apply a first cathodic potential to a first workingelectrode and a first anodic potential to a second working electrode,the first cathodic and anodic potentials corresponding to thoserespective potentials necessary to establish current flow through thesample due to diffusional recycling of the first redox reversiblespecies. Optionally the potential on one working electrode can be set ata first diffusible species dependent, anodic potential and current flowis measured as the potential of the other working electrode is sweptthrough a potential corresponding to the predetermined diffusiblespecies dependent cathodic potential (or vice versa).

The cathodic and anodic potentials appropriate for each reversible redoxspecies can be readily determined by empirical measurement. The multipleredox reversible species used in performance of the method of thisinvention are selected to have redox potentials differing by at least 50millivolts, more preferably at least 100 millivolts, more preferably atleast 200 millivolts, from that of each other redox reversible speciesutilized in the method. The difference in redox potentials of the redoxreversible species being used allow each species to be detected withoutsignificant interference from the second or any other redox reversiblespecies in the liquid sample. A steady state current flow is rapidlyestablished at each of the working electrodes following application ofthe anodic and cathodic potentials. Current flow can be measured ateither or both working electrodes, and it is proportionate to theconcentration of the recycling redox reversible species.

Second cathodic and anodic potentials are applied to the workingelectrodes wherein said second potentials correspond to those respectivepotentials necessary to establish current flow through the sample due todiffusional recycling of the second redox reversible species withoutsignificant interference from the first redox reversible species, andthe resulting steady state current flow is measured. This step isrepeated for each redox reversible species utilized in the method. Themeasured current flows are then correlated to known concentrations ofthe respective diffusible redox reversible species. Those concentrationsare proportionate to the respective analyte concentrations.

The method steps can be conducted using a programed bipotentiostat tocontrol potentials on the electrode structure in contact with thesample. The bipotentiostat can be included either in a desktop orhand-held meter further including means for reading values for steadystate current, storing said values, and calculating analyteconcentrations using a microprocessor programmed for making suchcalculations.

The redox reversible species utilized in the method comprise aliquid-sample-diffusible conjugate of a ligand analog of an analyte inthe liquid sample and a redox reversible label. The term “ligand analog”as used in defining the present invention refers to a chemical speciescapable of complexing with the same specific binding partner as theanalyte being measured and can include the analyte itself, provided thatthe molecular weight of the conjugate is less than about 50,000, morepreferably less than about 10,000 Daltons. Most preferably the molecularweight of the conjugate of the ligand analog and the redox reversiblelabel is between about 500 and about 5,000 Daltons. Low molecular weightredox reversible species are most desirable in view of thediffusion-based electrochemical detection technique utilized in carryingout the present method.

The term “redox reversible label” as used herein refers to a chemicalspecies capable of reversible oxidation and reduction in a liquidsample. It can be in the form of an organic moiety, for example, achemical group comprising a nitrosoaniline, a catechol, hydroquinone, oran aminophenol group. Alternatively, the redox reversible label can bean inorganic or organometallic species capable of undergoing reversibleoxidation and reduction in a liquid sample. Such species may be, forexample, complete molecules, portions of molecules, atoms, ions, or moreparticularly, ion complexes. Redox reversible labels are well-known inthe art and include ligand complexes of transition metal ions, forexample iron (ferrocene and ferrocene derivatives), ruthenium andosmium.

The relative amounts of the first and second redox reversible speciesand the respective specific binding partners for the targeted analytesto be measured in the method can be determined empirically. They aredependent on the concentration ranges of the targeted analyte, and thebinding stoichiometry of the specific binding partner, the bindingconstant, the analyte and the corresponding redox reversible species.The amounts of each reagent appropriate for each analyte being measuredcan be determined by empirical methods.

The redox reversible species typically comprises a conjugate of a ligandanalog of an analyte in a liquid sample and a redox reversible label.The conjugate is prepared by linking the ligand analog to the labeleither covalently through bifunctional linking agents or by combinationof covalent linkages and art-recognized specific binding entities (forexample, biotin-avidin).

In one embodiment of the invention the specific binding partner for eachanalyte is an antibody and the ligand analog is selected so that itbinds competitively with the analyte to the antibody. There are,however, other examples of ligand-specific binding partner interactionsthat can be utilized in developing applications of the present method.Examples of ligands and specific binding partners for said ligands arelisted below.

Ligand Specific Binding Partner Antigen (e.g., a drug substance)Specific antibody Antibody Antigen Hormone Hormone receptor Hormonereceptor Hormone Polynucleotide Complementary polynucleotide strandAvidin Biotin Biotin Avidin Protein A Immunoglobulin ImmunoglobulinProtein A Enzyme Enzyme cofactor (substrate) Enzyme cofactor (substrate)Enzyme Lectins Specific carbohydrate Specific carbohydrate Lectins oflectins

The term “antibody” refers to (a) any of the various classes orsubclasses of immunoglobulin, e.g., IgG, IgM, derived from any of theanimals conventionally used, e.g., sheep, rabbits, goats or mice; (b)monoclonal antibodies; (c)intact molecules or “fragments” of antibodies,monoclonal or polyclonal, the fragments being those which contain thebinding region of the antibody, i.e., fragments devoid of the Fc portion(e.g., Fab, Fab¹, F(ab′)₂) or the so-called “half-molecule” fragmentsobtained by reductive cleavage of the disulfide bonds connecting theheavy chain components in the intact antibody. The preparation of suchantibodies are well-known in the art.

The term “antigen” used in describing and defining the present inventionincludes both permanently antigenic species (for example, proteins,peptides, bacteria, bacteria fragments, cells, cell fragments, drugsubstances, and viruses) and haptans which may be rendered antigenicunder suitable conditions.

In one embodiment of the invention there is provided a method formeasuring two proteinaceous analytes in a liquid sample wherein theligand analog component of the first redox reversible species is apeptide comprising an epitope of a first analyte and the ligand analogcomponent of a second redox reversible species is a peptide comprisingan epitope of a second analyte. One specific binding partner utilized inthe method is an antibody recognizing the epitope of the first analyte,and the other specific binding partner is an antibody recognizing theepitope of the second analyte. In another application of the presentmethod two independent measurements are performed on a single analyte ina liquid sample. In that embodiment the respective ligand analogcomponent of the first and second redox reversible species are differentligand analogs of the targeted analyte. Where the targeted analyte is aproteinaceous compound, the ligand analog component of the first redoxreversible species is a peptide comprising a first epitope of theanalyte, and the ligand analog of the second redox reversible species isa peptide comprising a second epitope of the analyte, and the specificbinding partners are first and second antibodies, each recognizingrespective first and second analyte epitopes.

In one preferred embodiment of the invention, at least one of the redoxreversible labels is an osmium complex. In another embodiment, bothredox reversible labels are osmium complexes, each having an oxidizingpotential difference of at least 50, most preferably at least 200millivolts. Illustrative of the redox reversible osmium complexes foruse in this invention are complexes of the formula

wherein

R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,1 0-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group,

R and R₁ are coordinated to Os through their nitrogen atoms;

q is 1 or 0;

R₇ is B-(L)_(k)—Q(CH₂)_(i)—;

R₂ is hydrogen, methyl, or ethyl when q is 1, and R₂ isB—(L)_(k)—Q(CH₂)_(i)—when q is 0;

wherein in the group B—(L)_(k)—Q(CH₂)_(i—)Q is O,S, or NR₄ wherein R₄ ishydrogen, methyl or ethyl;

—L—is a divalent linker;

k is 1 or 0;

i is 1, 2, 3, 4, 5 or 6; and

B is hydrogen or a group comprising a ligand capable of binding to aspecific binding partner;

Z is chloro or bromo;

m is+1 or+2;

X is a mono- or divalent anion, e.g., chloride, bromide, iodide,fluoride, tetrafluoroborate, perchlorate, nitrate, sulfate, carbonate,or sulfite;

Y is monovalent anion, e.g., chloride, bromide, iodide, fluoride,tetrafluoroborate, perchlorate or nitrate; and

n is 1 or zero,

provided that when X is a divalent anion, n is zero,

and when m is 1, n is zero and X is not a divalent anion.

Another redox reversible osmium complex for use in the present method isa compound of the formula

wherein

R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group,

R₅ is 4-substituted-2,2′-bipyridyl or 4,4′-disubstituted-2,2′-bipyridylwherein the substituent is the group B—(L)_(k)—Q(CH₂)_(i)—and the4′-substituent is a methyl, ethyl or phenyl group;

R, R₁ and R₅ are coordinated to Os through their nitrogen atoms;

Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl or ethyl;

—L—is a divalent linker;

k is 1 or 0;

i is 1, 2, 3, 4, 5 or 6;

B is hydrogen or a group comprising a ligand capable of binding to aspecific binding partner;

d is+2or+3;

X and Y are anions selected from monovalent anions, e.g., chloride,bromide, iodide, fluoride, tetrafluoroborate, perchlorate, and nitrateand divalent anions, e.g., sulfate, carbonate or sulfite wherein x and yare independently 0, 1, 2, or 3 so that the net charge of X_(x)Y_(y)is−2or−3.

Redox reversible conjugate species of each of those formulas areprepared from the corresponding compounds wherein K is O and B ishydrogen by reacting such compounds with either a hetero functionalcrosslinker of the formula S—L′—T wherein L′ is a divalent linker and Sand T are different electrophilic groups capable of reacting with anucleophilic group to form a covalent bond, or with a homofunctionalcrosslinker of the formula S—L′—T wherein L′ is a divalent linker and Sand T are the same electrophilic groups capable of reacting with anucleophilic group to form a covalent bond. The resulting products arethen reacted with ligand analogs using classical coupling reactionconditions to product the conjugate species. The oxidizing potentials ofthe respective bis(bipyridyl) and tris(bipyridyl) osmium complexesdefined above is such that the respective complexes can be used asreversible redox labels for the respective redox reversible species inperformance of the method. FIG. 8 illustrates a cyclic voltammogram fora liquid sample containing equimolar (50 μM) amounts of a bis(bipyridyl)imidazolyl chloroosmium complex and a tris(bipyridyl) osmium complex.

In another embodiment of the invention there is provided a device fordetecting or quantifying one or more analytes in a liquid sample. Thedevice comprises

at least two redox reversible species, each capable of diffusion in saidliquid sample at least in the presence of a respective predeterminedanalyte, said redox reversible species having respective redoxpotentials differing by at least 50 millivolts,

an electrode structure for contact with the liquid sample in saidchamber, said electrode structure including a reference electrode andworking electrodes dimensioned to allow diffusional recycling of adiffusible redox reversible species in a liquid sample in contact withthe electrode structure when a predeterminedredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to a second working electrode, saiddiffusional recycling of said species being sufficient to sustainmeasurable current through each working electrode, and

conductors communicating with the respective electrodes for applyingsaid anodic potential and said cathodic potential and for carrying thecurrent conducted by the electrodes.

The device can be constructed using procedures and techniques that havebeen previously described in the art for construction of biosensorsemploying electrometric detection techniques. Thus, for example, thedevice can include a chamber that has a receiving port, and the chamberis dimensioned so that it fills by capillary flow when the liquid sampleis contacted with the sample receiving port. The electrode structure canbe formed on a plate that defines a wall of the chamber so that theelectrode structure will contact a liquid sample in the chamber. Thus,for example, the device can be constructed using the general proceduresand designs described in U.S. Pat. No. 5,141.868, the disclosure ofwhich is expressly incorporated herein by reference. The features of thepresent invention can also be incorporated into other electrochemicalbiosensors or test strips, such as those disclosed in U.S. Pat. Nos.5,120,420; 5,437,999; 5,192,415; 5,264,103; and 5,575,895, thedisclosures of which U.S. Pat. are expressly incorporated herein byreference. The device can be constructed to include the predeterminedamounts of the redox reversible species and the specific bindingpartners. For example, a mixture of such reagents can be coated onto awall of the sample chamber in said device during device construction, sothat the liquid sample is contacted with the reagent mixture as it isdelivered into the chamber for containing the sample. In one embodimentthe device is constructed for quantifying a first analyte and a secondanalyte in liquid sample. The device comprises two redox reversiblespecies, a first redox reversible species comprising a conjugate of aligand analog of the first analyte and a second redox reversible speciescomprising a conjugate of a ligand analog of the second analyte, and aspecific binding partner for each analyte so that each of said analyteanalog conjugates are capable of binding competitively with itsrespective analyte to a specific binding partner.

In one device embodiment of this invention the device further comprisesa bipotentiostat in electrical communication with the conductors forapplying a redox-reversible-species-dependent-cathodic potential to oneworking electrode and a redox-reversible-species-dependent-anodicpotential to a second working electrode. The biopotentiostat can beprogrammed to apply a sequence of potentials to the respective workingelectrodes. More particularly, the bipotentiostat can be programmed toapply first cathodic potential to a first working electrode and a firstanodic potential to a second working electrode, said first anodic andcathodic potentials corresponding to those potentials necessary toestablish current flow to the sample due to diffusional recycling of thefirst redox reversible species. The bipotentiostat is also programmed toapply a second cathodic potential to said first working electrode and asecond potential to the second anodic electrode, said second cathodicand anodic potentials corresponding to those potentials necessary toestablish current flow through the sample due to diffusional recyclingof the second redox reversible species. In an alternate embodiment thedevice includes first and second redox reversible species, and at leastfirst and second electrode structures for contact with the liquid samplein the chamber, each of said electrode structures comprising amicroarray of working electrodes, and means for switching thebipotentiostat between the first and second electrode structures. Inpreferred device embodiments there is provided means for measuringcurrent flow through the sample at each of the first and secondpotentials and preferably storing values for said current flows in aregister coupled to a microprocessor programmed to calculate analyteconcentrations based on said values.

In still another embodiment of the present invention there is provided akit for measuring the concentration of one or more analytes in liquidsample. The kit comprises

at least two redox reversible species for contact with the liquidsample, each capable of diffusion in the liquid sample at least in thepresence of a predetermined analyte, at least one of such species beinga conjugate of a ligand analog of an analyte and a redox reversiblelabel, said redox reversible species having respective redox potentialsdiffering by at least 50 millivolts;

a specific binding partner for each analyte;

an electrode structure for contact with the liquid sample, saidelectrode structure including a reference electrode and workingelectrodes dimensioned to allow diffusional recycling of diffusibleredox reversible species in the sample when a predeterminedredox-reversible-species-dependent-cathodic potential is applied to oneworking electrode and a predeterminedredox-reversible-species-dependent-anodic potential is applied to thesecond working electrode, said diffusional recycling of said speciesmeans sufficient to sustain a measurable current through the sample; and

conductors communicating with the respective electrodes for applyingsaid anodic potential and said cathodic potential and for carrying thecurrent conducted by the electrodes.

In one embodiment, the redox reversible species are mixed as a novelcomposition for contact with the liquid sample. In another embodimenteach of the redox reversible species and the specific binding partnerfor each analyte is mixed as a novel composition for contact with theliquid sample. Preferably, the redox reversible label of at least one ofthe redox reversible species comprises an osmium complex.

Preparation of Os Mediator Labels

The Os mediator bis(bipyridyl) imidazolyl chloroosmium has been shown tobe an excellent electron mediator for many oxido-reductase enzymes (U.S.Pat. No. 5,589,326). It has fast mediation kinetics (about 500 timesfaster than ferricyanide with glucose oxidase) and a relatively lowredox potential (+150 mV vs. Ag/AgCl). It has also very fast electrontransfer rate at electrode surface. More importantly, the organicligands on Os mediator can be functionalized so that it can becovalently linked to other molecules without detrimental effects onredox properties of the Os center. These unique properties of Osmediator make it an ideal electrochemical indicator for sensors based onimmunoaffinity.

Os mediators with these new ligands were synthesized using the sameprocedure used for Os free mediator. Their synthesis consists of twomajor process steps as outlined below. Details of these processing stepsare described below.

The first process step involves the synthesis of Os intermediate,cis-bis(2,2′-bipyridyl) dichloroosmium(II), from commercially availableosmium salt using the following scheme. The intermediate product isisolated through recrystallization in an ice bath.

K₂Os^(IV)Cl₆+2bpy ^(DMF) [Os^(III)(bpy)₂Cl₂]Cl+2KCl

2[Os^(III)(bpy)₂Cl₂]Cl+Na₂S₂O₄+2H₂O ^(0° C.)

2Os^(II)(bpy)₂Cl₂↓+2Na⁺+2SO₃ ⁼+4H⁺+2Cl

The second process step involves the reaction between Os intermediateand histamine or 4-imidazoleacetic acid (or a substituted bipyridine forpreparation of the tris(bipyridyl) complexes) to produce Os mediatorswith the appropriate “handle”. The desired product is then precipitatedout from solution by addition of ammonium tetrafluoroborate.${{{Os}^{II}({bpy})}_{2}{Cl}_{2}} + {{histamine}\quad {\frac{{EtOH}\text{/}H_{2}O}{\Delta}\quad\lbrack {{{Os}^{II}({bpy})}_{2}({histamine})} \rbrack}{Cl}}$$\quad \begin{matrix}{ {{\lbrack \quad {{Os}^{II}({bpy})_{2}\quad ({histamine})\quad {Cl}} \rbrack \quad {Cl}} + \quad {{NH}_{4}\quad {BF}_{4}}}arrow \quad} \\{\quad \lbrack \quad {{{Os}^{II}({bpy})}_{2}\quad  {(\quad {histamine})\quad {Cl}}\quad \rbrack \quad  {BF}_{4}\quad\downarrow\quad {+ {NH}_{4}} {Cl}} }\end{matrix}$

These Os mediators can also be easily converted to oxidized form, i.e.Os(III) using nitrosonium tetrafluoroborate. However, this isunnecessary since the Os revert back to reduced form anyway at alkalineconditions during conjugation reactions. And it does not requireoxidized form of Os(III) for the detection on the biosensor.

A. Simple Mixed Mediator Measurement

1. Interdigitated array microelectrodes (IDA) are produced throughphotolithographic means by art-recognized methods, (See W 97/34140; EP0299,780; D. G. Sanderson and L B. Anderson, Analytical Chemistry, 57(1985), 2388; Koichi Aoki et al., J. Electroanalytical Chemistry, 25691988) 269; and D. Niwa et la., J. Electroanalytical Chemistry, 167(1989) 291. Other means which are standard in lithographic processingmay also be used to produce the desired patterns of a conductor oninsulator substrate.

2. Reversible mediators are selected from those described herein andthose described references (U.S. Pat. Nos.4,945,045 and 5,589,325, thedisclosures of which are incorporated herein by reference). Preferablytwo different mediators are selected with potentials which differ by atleast 100 mV, more preferably at least 200 mV. Examples of suitablemediators include the Os(bipy)ImCl described herein and in U.S. Pat. No.5,589,326, the disclosure of which is incorporated herein by reference,and ferrocene, described in U.S. Pat. No. 4,945,045 and EP 0142301, thedisclosures of which are incorporated herein by reference. Mixtures ofthese mediators are made in aqueous solution, for examplephosphate-buffered saline (PBS). Concentrations between about 1 uM and1000 uM may conveniently be measured.

3. The IDA is connected to a bipotentiostat, an instrument capable ofcontrolling the potential of two separate electrodes. Also provided is areference electrode. This non-polarizable electrode serves as thereference for the two applied potentials and may also serve as thecounter electrode. Any non-polarizable electrode may be used, forexample Ag/AgCl, such as may be obtained from ABI/Abtech. An auxiliaryelectrode can also be used for controlling current flow through theworking electrodes. The mixtures are placed on the IDA electrode and thereference electrode also contacted with the mixture, or the IDA alongwith the reference electrode may be dipped into the mixture.

4. To measure Mediator 1 (Os(bipy)21mCl) A cathodic potential is appliedto one set of fingers of the IDA which is capable of reducing mediator 1(ca−50 mV vs. Ag/AgCl). An anodic potential is applied to the other setof fingers of the IDA which is capable of oxidizing mediator 1 but notmediator 2 (or any other mediators) (ca 250 mV vs Ag/AgCl). After ashort time (msec to sec), a steady state current will be measurablewhich is dependent only on the concentration of mediator 1.

5. To measure Mediator 2 (Ferrocene) A cathodic potential is applied toone set of fingers of the IDA which is capable of reducing mediator 2but not mediator 1 (ca 250 mV vs. Ag/AgCl). An anodic potential isapplied to the other set of fingers of the IDA which is capable ofoxidizing mediator 2 (ca 550 mV vs Ag/AgCl) . After a short time (msecto sec), a steady state current will be measurable which is dependentonly on the concentration of mediator 2.

Specific Binding Assay with Mixed Mediator Measurement Specific Assay ofHbAlc in a Blood sample

1. IDA electrodes are provided as in Paragraph A above.

2. Conjugates of mediators 1 and 2 and haptens or specific bindingmembers are provided using art-recognized procedures for covalentcoupling using either a homo-functional or hetero-functional linker.Specifically, a synthetic peptide corresponding to the N-terminalsequence of the β-chain of HbAlc is conjugated to the osmium complex.Similarly, a synthetic peptide corresponding to the N-terminal sequenceof HbA0 is conjugated to a second mediator, for example ferrocene.

3. Antibodies for the analytes (HbAlc and HbA0) which react specificallywith the N-terminal peptides which have been incorporated into theconjugate are provided by standard methods for producing polyclonalantibodies. In this case, sheep were immunized with carrier proteins towhich were conjugated the synthetic peptide sequences for HbAlc andHbA0. Following the appropriate immunization schedule, the sheep werebled, and the antibody isolated from the blood via ion exchangechromatography, followed by immunosorbent purification on a column ofthe same N-terminal peptide with a different linker.

4. Appropriate stoichiometry of the reaction was determined for the tworeactions independently by methods standard for immunoassay development.A solution containing a fixed amount of labeled conjugate was mixed witha solution with varying amounts of antibody, and, following anappropriate incubation period, the amount of free conjugate remainingwas measured on the IDA electrode using the procedure described above.The amount of antibody just sufficient to achieve maximum inhibition ofthe conjugate (ca>80%) was selected.

5. Reagent solution 1 was made containing a mixture of the twoconjugates in the appropriate concentrations. Reagent solution 2 wasmade containing a mixture of the two antibodies in the amountsdetermined above. A blood sample was diluted ca 20-fold in a solution of25mM citric acid/0.5% Brij-35. Following a 30 second incubation to allowfor lysis and denaturation of the hemoglobin, to 66 uL of this dilutedsample was added 33 uL of 1 M phosphate buffer, to adjust the pH back toneutral. 30 uL of antibody solution 2 was added, and the mixture allowedto incubate 30 sec. Then 30 uL of conjugate solution 1 was added, andthe mixture measured on the IDA electrode. The concentration of HbAlc inthe sample is related to the current measured from Mediator 1, and theconcentration of HbA0 is related to the current from Mediator 2. The%HbAlc in the sample is related to the ratio of the measured amounts ofMediator 1 and Mediator 2.

Application to HbAlc Assay

Hemoglobin Alc is a specific glycohemoglobin in which the glycosylationtakes place at the N-terminal of hemoglobin β-chain. The antibody bindsspecifically to HbAlc has an epitope sequence of Gluc-Val-His-Leu-Thr.To facilitate conjugation to other molecules, a nonnative amino acid hasbeen added to the sequence, e.g., Cys, Lys, or Lys-MH, to produce Alcpeptides including: 1) Gluc-Val-His-Leu-Thr-Lys-MH; 2)Gluc-Val-His-Leu-Thr-Lys; 2) Gluc-Val-His-Leu-Thr-Cys.

HbAlc assay requires measuring both Al c concentration and totalhemoglobin concentration and reports the results as a ratio of these twomeasurements (% HbAlc). It is advantageous to assay both Alc and totalhemoglobin using same principle because ratioing would minimize biasesdue to environmental effects. Thus antibody has been raised to bindspecifically to hemoglobins with unglycosylated N-terminus, i. e. withan epitope sequence of Val-His-Leu-Thr. Similarly, nonnative amino acidis added to the sequence to facilitate conjugation. The peptides usedfor total hemoglobin measurement is termed as AO peptide. AO peptidesthat have been used in the preparation of Os mediator-peptide conjugatesinclude Val-His-Leu-Thr-Cys and Val-His-Leu-Thr-Lys.

Conjugation Chemistry and Conjugates

There are many types of conjugation chemistry that can be employed tolink Os mediator to a peptide. The following two conjugation chemistriesemployed for the preparation of Os mediator-peptide conjugates have alsobeen commonly used for preparing protein conjugates: 1) formation ofamide bond by reactive ester with primary amine; 2) formation ofthioether bond by maleimide with sulfhydryl group. Amide bond ispreferred over thioether bond because amide bond is generally morestable. Based the preferred conjugation chemistry, the ligand on Osmediator can be functionalized with either a primary amine group or acarboxylic acid group. The best location for these functional groups isbelieved to be the C-4 or C-5 positions on the imidazole ligand of Osmediator, however, functionalization through the non-Os-complexedimidazole ring nitrogen atom can also be carried out. Two differentfunctionalized Os mediators were synthesized as described above.

Os mediator (a) was formed with histamine while Os mediator (b) wasformed with imidazolacetic acid. However, it was found that the iminonitrogen of the imidazole ring interferes with the activation ofcarboxylic acid group to reactive ester (i.e., N-hydroxysuccinimideester) using carbodiimide. Thus, use of carboxylic acid functionalizedOs mediator in the synthesis of Os mediator-peptide conjugates gave muchless favorable results.

The amine group on histamine ligand of Os mediator readily reacts withN-hydroxysuccinimide (NHS) ester to form amide bond. Two types ofcrosslinkers have been employed to link Os mediator to peptides, (a)heterofunctional crosslinker, having a NHS ester at one end and theother end has a maleimide or a sulthydryl group; and (b) homofunctionalcrosslinker, e.g. both ends have NHS esters.

In the case of heterofunctional crosslinker, the crosslinker is firstreacted with Os mediator with histamine ligand (Os histamine) at 1:1molar ratio. One particular point needs to be noted here. Os mediator inoxidized form, i.e. Os(III), can instantly oxidize sulfhydryl group toform disulfide bond. It is important to keep Os center in the reducedform by addition of a small amount of reductant such as sodiumdithionite during the conjugation processes. The reaction progress canbe monitored by analytical reverse-phase HPLC on a C 18 column. Then theOs mediator-crosslinker adduct is isolated via preparative HPLC and thecollected fraction is subsequently freeze-dried. Finally, the Osmediator-crosslinker adduct is reacted with the appropriate peptide toform Os mediator-peptide conjugate. Again, the product is isolated bycollecting appropriate fraction in preparative HPLC and the collectedfraction is then freeze-dried.

Two different heterofunctional crosslinkers have been used for thesynthesis of Os mediator-peptide conjugates. SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) is used forcystein-containing peptide, while SATA (N-succinimidylS-acetylthioacetate) is used for maleimide-containing peptide. Three Osmediator-peptide conjugates (two with Alc peptide and one with A0peptide) have been made using heterofunctional crosslinkers and theirstructure are shown below: (a) Os-SMMC-A1c; (b) Os-SATA-A1c, and (c)Os-SATA-AO.

However, it has been found that these conjugates were not stable whenthey were stored as solutions. Analytical HPLC results indicated thatthese conjugates degrade. Mass spectroscopy confirmed that theinstability is due to splitting of thioether bond present in theseconjugates.

In order to avoid thioether bond in the conjugate, homofunctionalcrosslinker containing two NHS esters was used instead to prepare theconjugates. The crosslinker used was DSG (disuccinimidyl glutarate). Inorder to prevent the formation of crosslinked Os mediator, i.e.Os-crosslinker-Os, a large excess of homofunctional crosslinker was usedin the reaction with Os histamine at 4:1 molar ratio. Under thiscondition, only the desired product, i.e. Os-crosslinker, was formed.The Os-DSG-Alc conjugate was similarly prepared using the proceduredescribed earlier.

The preparation of analogous Os-DSG-A_(o)conjugate requires and extrastep since the unglycated N-terminal amine of HbAA_(o) peptide is alsoreactive toward NHS ester. In this case, the N-terminal amine of HbA_(o)peptide is first protected by either a base-labile Fmoc¹ or anacid-labile Boc² group. After reacting with Os-DSG adduct to formOs-DSG-A_(o) conjugate, the protecting group is then cleaved usingappropriate deprotection method (adding base for Fmoc or acid for Boc).The peptides prepared by solid-phase peptide synthesis already haveN-terminal Fmoc protecting groups. The protecting groups are usuallyremoved prior to cleavage of peptide from the resin beads, but they canalso be left on if so desired. The HbA_(o) peptide from Zymed has anintact Fmoc protecting group at N-terminus. Using this strategy theOs-DSG-A_(o) conjugate was successfully synthesized.

¹Fmoc=9-flourenylmethyloxycarbonyl

²Boc =t-butyloxycarbonyl

Many analytes cannot be assayed using enzyme-based sensors. They requirethe development of affinity biosensors or immunosensors which are basedon the selective binding of antigens to antibodies. The key to thedetection of this binding event on electrochemical sensors is theinclusion of antigens labeled with redox labels.

Bis(2,2′-bipyridyl) imidazolyl chloroosmium, a.k.a. Os mediator,possesses many properties that make it an excellent redox label for thispurpose. In addition, a “handle” for linking it covalently to antigenscan be added on without affecting its redox properties.

Several assay schemes can be used in affinity biosensors including, i)competitive binding assay (labeled antigen is competing for a limitednumber of binding sites); ii) sequential binding assays (labeled antigenis bound to excess binding sites); iii) heterogeneous assay (uses aseparation step to separate bound and free labeled antigens); and iv)homogeneous assay (no separation step). The steps involved in ahomogeneous sequential binding assay include binding the analyte to anantibody. The labeled antigen (analyte analog) binds to the remainingbinding sites on the antibody. Finally the leftover free labeled antigenis detected at electrode surface. The resulting current will be afunction of the amount of analyte present.

The detection of free labeled antigens can be achieved using eitherdirect detection or amplified detection methods. Direct detectionrequires the use of advanced electrochemical techniques such as acvoltammetry, differential pulse voltammetry, square wave voltammetry orac impedance in order to reach a sensitivity of 5 μM or less. Amplifieddetection methods use dc amperometry with amplification through reactionwith enzyme or chemical reductants or by using interdigital array (IDA)microelectrodes. The preferred detection method is amplified amperometrythrough cycling of free Os mediator label by using IDA microelectrodesin accordance with this invention.

General Analytical HPLC Method For Osmium Conjugates

All HPLC analysis were performed using a Beckman System Gold HPLC systemconsists of a 126 pump module and a 168 diode array detector module.

Stationary phase is a Vydac analytical reverse-phase C18 analyticalcolumn. Other parameters are listed below.

Mobil Phase: A=0.1%TFA³ in H₂O

B=0.1% TFA in CH₃CN

Flow rate: 1 mL/min

Gradient: 0−5 min: 10% B

5-45 min: 10% B−>50% B at 1%/min

45-50 min: 100% B

Detector: Channel A at 384 nm

Channel B at 220 nm.

³TFA=trifluoroacetic acid.

Synthesis of Bis(2,2′-bipyridyl) dichloroosmium

1. Charge a 1 L one-neck RB flask with 19.335 grams K₂OsCl₆ (0.04019mole) and 13.295 grams 2,2′-dipyridyl (0.08512 mole). Add 400 mL DMF todissolve all reactants.

2. Heat the solution to reflux and then maintain reflux for 1 hour. Thenturn off the heat and let solution cool to 30-40° C. at ambient.

3. Filter the reaction mixture using a medium grade glass-frit filter.Rinse the flask with additional 20 mL DMF and wash the filter.

4. Transfer the filtrate into a 3-L beaker. Charge another 2-L beakerwith 22.799 grams of NaS204 and dissolve in 2 L deionized water. Addthis solution to the beaker containing Os/DMF filtrate dropwise using adropping funnel. Keep the solution stirring at all time.

5. Then cool the mixture in an ice bath for at least 3 hours. Add ice asnecessary.

6. Filter the mixture “cold” using a ceramic filter with filter paper.Wash the content on the filter with 50 mL, water twice and 50 mL ethertwice.

7. Dry the product under high vacuum at 50° C. overnight (at least 15hours). Weigh the product and transfer into a brown bottle. Store in adesiccator at room temperature.

Typical yield=16 gram or 70%.

Product is analyzed by UV-Visible spectroscopy and elemental analysis.

UV-Vis: Peak λ (nm) ε (M⁻¹cm⁻¹) 382 10,745 465  9,914 558 11,560 836 3,873 EA: C % H % N % Cl % Os % H₂O % Theoretical 41.89 2.81 9.77 12.3633.17 0 Actual 40.74 2.92 9.87 11.91 0.41

Synthesis of Bis(2,2′-bipyridyl) histamine chloroosmium

1. Charge a 2L one-neck RB flask with 11.3959 gram Os(bpy)₂Cl₂ (0.0198mole) and 4.9836 gram histamine (0.0448 mole). Add 500 mL ethanol todissolve the reactants. Then add 250 mL deionized water.

2. Heat the solution to reflux and maintain reflux for 6 hours. Letsolution cool to RT at ambient.

3. Remove all ethanol using rotary evaporation. Then transfer thesolution into a 500 mL beaker. Dissolve 2.136 gram NH ₄BF ₄ in 20 mLwater. Add dropwise to Os solution. Precipitate forms. Cool in an icebath for 30 min. Filter the mixture using a ceramic filter with filterpaper. Wash the content on filter with ˜20 mL water twice. 4. Dry underhigh vacuum at 50° C. overnight (at least 15 hour). 5. Weigh the productand transfer to a brown bottle. Store in a desiccator at roomtemperature.

Typical yield=7.6 gram or 52%.

Product is analyzed by UV-visible spectroscopy and HPLC.

UV-Vis: Peak λ (nm) ε (M⁻¹cm⁻¹) 355 7,538 424 7,334 514 7,334 722 2,775

HPLC: Elutiontime=18.0 min

Purity by HPLC range from 65-85%

Preparation of [Os(bpy)₂(histamine)Cl]-heterofunctional crosslinkeradduct

1. Weigh 0.1167 g [Os(bpy)₂(histamine)Cl] BF₄ (0.162 mmol) and transferto a 5 mL Reacti-Vial. Add 1.0 mL DMF to dissolve the reactant. Add 25μL triethylamine.

2. Add 0.0508 g SMCC(0.150 mmol)or 0.0390 g SATA (0.168 mmol). Stir thereactants at RT for 2 hours. Inject a sample into HPLC to monitorreaction progress.

3. If reaction is complete, dilute the solution with 0.1% TFA buffer toa final volume of 4.5 mL. Inject into preparative BPLC and collect theproduct peak.

4. Freeze dry the collected fraction overnight.

5. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=40 mg or 25%.

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC: Elution time = 32.1 min m⁺/e = 434.2 Os-SATA:Elution time = 27.5 min m⁺/e = 382.8

Preparation of [Os(bpy)₂(histamine)Cl ]-homofunctional crosslinkeradduct

1. Weigh 0.2042 g DSG (0.626 mmol) ) and transfer to a 5 mL Reacti-Vial.Add 0.75 mL DMF to dissolve the reactant.

2. Weigh 0.1023 g [Os(bpy)₂(histamine)Cl]BF₄ (0.142 mmol) and transferto a separate 5 mL Reacti-Vial. Add 1.0 mL DMF to dissolve the reactant.Add 25 μL triethylamine. Then add Os/DNF solution dropwise to DSG/DWsolution with constant stirring. After reacting for 2 hours at RT,inject a sample into HPLC to monitor reaction progress.

3. If reaction is complete, dilute the solution with 0.1% TFA buffer toa final volume of 4.5 mL. Inject into preparative HPLC and collect theproduct peak.

4. Freeze dry the collected fraction overnight.

5. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=45 mg or 35%.

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG: Elution time = 27.1 min m⁺/e = 429.2 and 859.6

Preparation of Os-SATA-Alc Conjugate

1. Weigh 40.5 mg Os-SATA (0.0529 mmol) and transfer to a 5 mLReacti-Vial with stir bar. Add 1.0 mL PBS (pH 7.5) to dissolve. Add 20mg Na₂S₂O₄ in order to keep Os in reduced form.

2. Add 1.0 mL deacetylation buffer (PBS pH7.5+0.5 M hydroxylamine and 25mM EDTA) to deprotect the sulfhydryl group. Inject a sample intoanalytical HPLC to determine whether deprotection is complete byappearance of a new peak at 25.8 min.

3. Add 45 ig HbAlc-MH peptide (0.0474 mmol) and let react at RT for 1hour. Inject a sample into analytical HPLC to monitor reaction progress.

4. If reaction is complete, dilute the mixture with 0.1 %TFA buffer to afinal volume of 4.5 mL. Inject into preparative HPLC to collect productpeak.

5. Freeze dry the collected fraction overnight (at least 15 hour).

6. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=12 mg

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SATA-Alc: elution time = 27.6 min m⁺/e = 559.1 and 838.5

Preparation of Os-SMCC-Alc Conjugate

1. Weigh 39.0 mg Os-SMCC (0.0452 mmol) and transfer to a 5 mLReacti-Vial with stir bar. Add 1.0 mL PBS (pH 6.0) to dissolve.

2. Add 30.0 mg Hblc-Cys peptide (0.0450 mmol). Let reaction proceed atRT for 2 hours. Inject a sample into analytical HPLC to monitor reactionprogress.

3. If reaction is complete, dilute the mixture with 0.1% TFA buffer to afinal volume of 4.5 mL. Inject into preparative HPLC to collect productpeak.

4. Freeze dry the collected fraction overnight (at least 15 hour).

5. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=12 mg

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC-Alc: elution time = 27.6 min m⁺/e = 480.5 and 534.4

Preparation of Os-SMCC-Ao Conjugate

1. Weigh 37.0 mg Os-SMCC (0.0426 mmol) and transfer to a 5 mLReacti-Vial with stir bar. Add 1.0 mL PBS (pH=6.0) to dissolve.

2. Add 24.3 mg HbAo-Cys peptide (0.0425 mmol). Let reaction proceed atRT for 2 hours. Inject a sample into analytical HPLC to monitor reactionprogress.

3. If reaction is complete, dilute the mixture with 0.1 %TFA buffer to afinal volume of 4.5 mL. Inject into preparative HPLC and collect theproduct peak.

4. Freeze dry the collected fraction overnight (at least 15 hour).

5. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=15 mg

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC-A_(o): elution time = 27.9 min m⁺/e = 360.7 and 720.5

Preparation of Os-DSG-AIc Conjugate

1. Weigh 32.0 mg Os-DSG (0.037 mmol) and transfer to a 5 mL Reacti-Vialwith stir bar. Add 0.75 mL DMF to dissolve. Add 25 μL triethylamine.

2. Add 26.5 mg HbAlc-Lys peptide (0.0349 mmol). Let reaction proceed atRT for 2 hours. Inject a sample into analytical BPLC to monitor reactionprogress.

3. If reaction is complete, dilute the mixture with 0.1%TFA buffer to afinal volume of 4.5 mL. Inject into preparative HPLC and collect theproduct peak.

4. Freeze dry the collected fraction overnight (at least 15 hour).

5. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=16 mg

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG-Alc: elution time = 23.5 min m⁺/e = 501.8 and 752.8

Preparation of Os-DSG-Ao Conjugate

1. Weigh 52.0 mg Os-DSG (0.0605 mmol) and transfer to a 5 mL Reacti-Vialwith stir bar. Add 1.0 mL DMF to dissolve. Add 25 μL triethylairiine.

2. Add 49.1 mg Fmoc-HbA₀ peptide (0.0606 mmol). Let reaction proceed atRT for 2 hours. Inject a sample into analytical HPLC to monitor reactionprogress by the appearance of peak at 40.3 min for Os-DSG-A_(o)(Fmoc).

3. If reaction is complete, inject additional 100 μL triethylamine.After I hour, inject sample into analytical HPLC to determine whetherall Fmoc protection group is removed by disappearance of the peak at40.3 min.

4. If removal of Fmoc is complete, dilute the mixture with 0. 1%TFAbuffer to a final volume of 4.5 mL. Inject into preparative HPLC tocollect product peak.

5. Freeze dry the collected fraction overnight (at least 15 hour).

6. Weigh the product and transfer to a brown bottle. Store in adesiccated bag at−20° C.

Typical yield=16 mg

Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG-A_(o): elution time = 23.2 min m⁺/e = 447.4 and 670.3

Synthesis of bis (4,4′-dimethyl-2,2′-bipyridyl) 4-methyl-4′-carboxylpropyl-2,2′-bipyridyl osmium [Os(dm-bpy)2(mcp-bpy)]Cl2

Potassium hexachloroosmium was reacted with 4,4′-dimethyl-2,2′-dipyridylat 1:2 molar ratio by refluxing in DMF. The potassium chlorideprecipitate was filtered and the dimethyl-bipyridyl dichloroosmiumcomplex was reduced from+3 oxidation state to+2 oxidation state usingexcess sodium dithionite. The product was recrystallized in DMF/watermixture at 0° and recovered by filtration.

4,4′-Dimethyl-2,2′-bipyridyl dichloroosmium was reacted with4-methyl-4′-carboxylpropyl-2,2′-dipyridyl by refluxing in ethyleneglycol. The solvent was removed by rotary evaporation. The product wasdissolved in DMF and precipitate in ethyl ether. The product was driedin a vacuum oven overnight.

Analyticals: Product and intermediate product were analyzed by HPLC andmass spectroscopy for purity and identity of the compound.

Os(dm-bpy)2Cl2: Theoretical MW =629.6, MS showed 8 isotope peaks withmost abundant peak at 630. HLPC elution time at 29.94 min with a purityof 90%+

Os(dm-bpy)2(mcp-bpy): MS confirmed the MW at 814.5 and HPLC showed apurity greater than 85%.

Synthesis of biotin-Os complex conjugate Biotin-Os(dm-bpy)2(mcp-bpy)]Cl2

The carboxyl group was activated by reacting the above Os complex withdicyclohexylcarbodiimide in the presence of N-hydroxysuccinimide. Theactive ester Os complex was isolated using preparative HPLC method andthen reacted with amine-containing biotin to form the final conjugate.

Experiment to Independently Measure the Concentration of TwoElectroactive Conjugate Species

Os(bipy)HisCl-DSG-HbAlc was prepared as described above.

Ferrocene-AMCHA-DADOO-biotin was prepared from ferrocene monocarboxylicacid, the crosslinker aminomethylcyclohexylic acid, the chain extender1,8-diamino-3,6-DiOxoOctane and biotin as described elsewhere.

Mixtures of the two conjugates were prepared to evaluate the ability ofthe method of the invention to independently measure the concentrationof the conjugates, and make corrections for variations in reagentamounts, electrode response, and environmental conditions.

Part 1: Simple Mixed Conjugate Response

The following matrix of solutions was prepared in 10 mM phosphate bufferwith 150 mM NaCl and 0.5 % Brij-35, a non-ionic surfactant.

Os-DSG- Ferrocene- Os-DSG- Ferrocene- HbAlc Biotin HbAlc Biotin uM/luM/l uM/l uM/l 0  0 0 12.5 6.25  0 6.25 12.5 12.5  0 12.5 12.5 25  0 2512.5 0 25 0 50 6.25 25 6.25 50 12.5 25 12.5 50 25 25 25 50

An Interdigitated array ( IDA) microelectrode was fabricated accordingto the procedures described. In addition to the IDA, the chip had asilver/silver chloride electrode on the surface to function as thereference electrode and counter electrode. This electrode was producedwith the same lithographic process, and then electroplated with silverand silver chloride according to standard techniques. The IDA wasconnected to a bipotentiostat capable of controlling the potentialrelative to the reference and measuring the current at each of theelectrodes of the IDA. Aliquots of the solutions were placed onto thesurface of the chip, such that the IDA and the reference electrodes werecovered.

Measurements were made by first applying−100 mV (vs. Ag/AgCl) to oneelectrode of the IDA, and 200 mV to the other electrode for a period of30 seconds. At this time, current was measured at each electrode. Then200 mV was applied to one electrode, and 550 mV to the other. After 30seconds, current was again measured. See FIG. 9 for a summary of theresults, which clearly demonstrate that the concentrations of the twomediators can be independently measured with this method.

Part 2. Concentration Co-variance of Dual Mediators on IDA Electrodes

In this experiment, it was demonstrated that by making a mixture ofknown concentrations of two different mediators, and measuring differentdilutions of that mixture by the method of the invention, the ratio ofthe concentrations of the mediators remains constant. ( Internalstandard application ).

The same two mediator conjugates were used as in Part 1. ( Os-DSG-Alcand Fc-Bi)

From a solution containing 40 uM of each conjugate, solutions containing27 uM, 32 uM, and 36 uM of each conjugate were prepared in the samebuffer ( PBS/Brij).

The solutions were measured as in the previous example. Each solutionwas measured on 5 different IDA electrodes. The results are presented asthe means, standard deviations, and coefficient of variation for eachsolution separately, and for all solutions pooled over all electrodes.

Individual Os-DSG- concentrations Alc Fc-Bi Os/FC 28 uM Mean 156 85 1.84S.D. 17.5 13.2 0.08 % C.V. 11.2 15.5 4.4 32 uM Mean 165 88 1.88 S.D. 117.2 0.04 % C.V. 6.7 8.2 2.0 36 uM Mean 189 102 1.85 S.D. 12.5 6.9 0.04 %C.V. 6.6 6.7 2.3 40 uM Mean 208 110 1.90 S.D. 9.5 5.5 0.06 % C.V. 4.65.0 3.4 Pooled Concentrations Mean 182 97 1.87 S.D. 24.4 13.2 0.06 %C.V. 13.4 13.5 3.4

This example clearly demonstrates that the internal standard effect ofmeasuring two conjugates or mediators and calculating the ratio givessignificantly improved precision of measurement, not only within eachsolution (compensation for variation between electrodes ) but over allsolutions (compensation for variation in sample dilution or amount).

Part 3. Temperature Compensation

It was desired to show the effectiveness of the method in compensatingfor environmental influences such as Temperature variation on theaccuracy or the measurement.

The same two conjugates were prepared in solution at 40 uM as before.They were measured as before on IDA electrodes, either at roomtemperature or warmed to 35-40° C. on a heated metal plate prior to themeasurement. The solutions were also warmed to 37° C. prior toapplication to the electrodes.

Room Temperature Warmed Ratio of response (23° C.) (35-40°) C. Warm/RTOs-DSG-Alc 261 387 1.45 Fc-Bi 179 268 1.5 Ratio Os/Fc 1.46 1.44 0.99

As demonstrated by the results, the measured values increase by almost50 % in the case of the warmed samples, which would lead to a largemeasurement error. However the use of the internal standard and ratiocalculation effectively eliminates the temperature dependence of theresult.

Immunoassay Detection of HbAic with Osmium Mediator Conjugates

The goal of all diabetic therapy is to maintain a near normal level ofglucose in the blood. Home blood glucose kits are available to monitorthe current glucose values and are valuable for diabetics to adjust dayto day insulin doses and eating habits. Unfortunately, since the testsonly measures a point in time result, it does not tell them the overalleffectiveness of their actions in maintaining glycemic control.Measurement of glycosylated hemoglobin is now becoming widely acceptedas an index of mean blood glucose concentrations over the preceding 6-8weeks and thus provides a measure of the effectiveness of a diabetic'stotal therapy during periods of stable control. Since monitoring adiabetic's glycated hemoglobin can lead to improved glycemic control,the ADA recommends routine measurements of four times a year up to oncea month for less stable type I diabetics.

Several technologies are available for the measurement of glycatedhemoglobin. They include immunoassays for HbAlc (TinaQuant, BMC;DCA2000, Ames; and Unimate, Roche), ion exchange (Variant, BioRad; EagleDiagnostics), and affinity chromatography (ColumrMate, Helena; GlyHb,Pierce).

One objective of this project is to develop a simple to use disposablestrip for electrochemical detection of HbAlc for use in both physicianoffices and the home.

The most significant parameter for assessing patient condition is ratioof HbAlc to HbA_(o), and thus the measurement of both glycated (HbAlc)and nonglycated (HbA0) values is required to calculate the ratio. Thisrequires two separate measurements. It is preferable to use the sametechnology to measure both the glycated and nonglycated fractions, thusremoving some sample and environmental interferences. Measurement ofHbAlc via electrochemical immunoassay is described below.Electrochemical HbA0 immunoassay measurements are carried out using thesame methods as that for HbAlc. The concentrations of HbA_(O) aresignificantly higher. One alternative to A₀ measurements usingimmunoaffinity would be to measure total hemoglobin directly usingbiamperometry or differential pulse voltammetry. This can be easilyaccomplished since hemoglobin is readily oxidized by[0s(bpy)₂(im)Cl]²⁺Cl ₂.

The N-terminal valine of the β-chain of hemoglobin A is the site ofglycosylation in HbAlc, and serves as a recognition site for theantibody. In whole blood the N-terminal valine is not accessible for theantibody to bind. Access is gained by lysing the red cells to releasethe hemoglobin followed by a conformation change (denaturing orunraveling) to adequately expose the HbAlc epitope. Dilution of thesample may occur as part of the lysing/denaturing process or may berequired post denaturing to prepare the sample for the antibody (adjustpH, other) or bring the sample into a range suitable for electrochemicalimmunoassay. In one embodiment, a fixed amount of antibody is incubatedwith the prepared sample and it binds to the HbAlc epitopes of thesample. The free antibody and the antibody bound sample is then combinedwith the osmium peptide conjugate (Alc or A₀) to allow the remainingunbound antibody to bind to the mediator label. When the mediator isbound to the antibody (a macromolecule), it can not freely diffuse tointeract with the electrode and thus currents generated aresignificantly reduced. The remaining unbound mediator label is thereforeproportional to the concentration of HbAlc in the sample. The unboundmediator can be measured electrochemically preferably using aninterdigitated array electrode with bipotentiostatic control.

Blood lysis is necessary to release the hemoglobin followed bydenaturing to expose the HbAlc epitope. Lysis can easily be accomplishedvia surfactants, osmotic effects of dilution with water, and directly bymany denaturants. Blood lysed through a freeze/thaw cycle was shown notto significantly interfere with the biamperometric measurement (“openrate” with and without lysed blood was almost identical). Conversely,denaturing the lysed blood with a variety of known denaturants to exposethe HbAlc epitope has shown significant suppression of theelectrochemical response, inhibiting measurement of an HbAlc doseresponse. Only LiSCN and citric acid from the list of evaluateddenaturants shown in Table 1 was able to expose the Alc epitope andminimize protein fouling enough to measure an HbAlc dose response.

Denaturing the sample for antibody recognition without severely foulingthe electrode surface is important for successful development of anHbAlc immunoassay. Although LiSCN has been used almost exclusively toshow feasibility, it has many limitations that would hinder its use inthe disposable. Citric acid, a solid at RT may offer benefits as adenaturant if it could be dried onto a strip followed by a diluent toadjust the pH to neutral. Acid or base blood denaturing followed by afinal pH adjustment with a buffered diluent is an area worth furtherevaluation. One problem that was initially encountered was precipitationin adjusting the pH back to neutral, which can be overcome by using adifferent buffer or with the addition of surfactants.

TABLE 1 Blood Denaturants Method Comments KSCN Initial work did not showa dose response with KSCN denatured blood. KSCN has a larger negativeeffect on the electrochemical response than LiSCN. Literature shows thatLiSCN is more effective than KSCN (concentrated efforts on LiSCN).DCA2000 Denaturing of blood not evident with the higher blood Bufferconcentrations required for this assay. Higher concen- trations of LiSCNare shown below. LiSCN Method used by Ames DCA2000 HbAlc immunoassay.Citric, Blood HbAlc dose response (high/low) was seen with Sulfuric, Hy-citric acid and was comparable to the response drochloric, with LiSCN &Evidence of blood denaturing was seen by Perchloric all: “solutionturned brown.” Citric acid is preferred. Acid Adjustment of pH toneutral after denaturing also saw problems of precipitation. Enzymemediated responses with Gluc-Dor at pH 5.7 reduces response 50% comparedto pH 7-8. Citric acid blood denaturing method is shown in FIG. 5.Pepsin/ Roche HbAlc immunoassay uses pepsin/citric acid to Citric Acidhemolyze and proteolytically degrade hemoglobin to glycoproteinsaccessible by the antibody. Denaturation was apparent by the colorchange to a brown- ish red solution. Hemoglobin Alc dose response(high/low) was obtained comparable to LiSCN and citric acid denaturants.The procedure was identical to that of citric acid used above with theexception of pepsin added to the acid. Results were identical to that ofcitric acid. TTAB (Tetra Method of denaturing used in the TinaQuantHbAlc decyl- turbidimetric immunoassay. trimethyl Evidence ofdenaturing: “solution turned green” ammonium TTAB concentrations0.0125-0.2% severely suppressed bromide) enzyme mediated(Glucdor/PQQ/Glucose) biamperometric measurements. Open rates were 16-50 nA compared to 140 nA without TTAB. NaOH Evidence of denaturing:“solution turned brown.” NaOH does not adversely effect the enzymemediated electrochemistry. Even at high pH the open rates do not change,although pH adjustment will probably be required to bring it within anoptimal range for the antibody. NaOH denatured blood suppresses the openrates probably due to protein fouling. Lowering the pH to neutral tendsto cause some precipitation.

TABLE 2 Effective Blood Denaturing Procedure for 2% Blood LiSCN (OneStep) LiSCN (Two Step) Citric Acid (2 Step) 40 μL 6 M LiSCN 960 μL 1.5 MLiSCN 200 μL 0.2 M Citric Acid 20 μL 5% Tween in 20 μL 5% Tween in PBS20 μL 5% Tween in PBS PBS 20 μL Blood 20 μL Blood 20μL Blood Mix(vortex) and Mix (vortex) and allow Mix (vortex) and allow to denatureto denature for 10 allow to denature for 10 minutes. minutes. for 10minutes. Dilute with 920 μL Dilute with 760 μL DI H₂O. 8X PBS (0.1%Tween) Denaturing time Denaturing time was not No optimization was notoptimized. optimized. studies were Limited data supports Data indicatesshorter performed. longer times for better times may be adequate.precision using this method. PBS = 10 mM Phosphate Buffer, 2.7 mM KCl,137 mM NaCl pH = 7.4 Increased level of surfactant (5% Tween) reduces oreliminates precipitate.

Electrode fouling caused by denatured blood proteins adsorbing to theelectrode surface can impede electron transfer and thus decreaseelectrode sensitivity.

Electrode fouling or passivation occurs more or less immediatelyfollowing sample contact with the surface thus minimizing the severityof denaturing in the sample should be the first approach. Surfaceconditions that are hydrophobic will favor adhesion of the proteins andthus fouling may be minimized with electrode surfaces of higher surfaceenergies. This explains why gold electrodes shows less fouling withdenatured blood than palladium. Reduction of protein fouling may beachieved by changing or protecting the electrode surface. Modificationsthat make the surface more hydrophilic should reduce the amount offouling and can be accomplished by argon or oxygen plasma treatment orcorona treatment. Selective coatings that could block the proteins fromreaching the electrode surface can usually partially circumvent theproblem have been used in the field to reduce fouling. Unfortunately,dramatic decreases in responses greater than seen with the denaturedblood proteins are normally noted with their use. Hydrophilic coatingssuch as PEO were also evaluated and showed some improvement, but havesimilar problems of decreased magnitude and precision caused by forcingreagents to diffuse through the polymers. Reagents dispensed and driedover the electrodes may help reduce the magnitude of protein foulingwith less negative effects.

Mediator concentration dose response, inhibition with antibody andreversal with a BSA-Alc polyhaptan were evaluated and summarized inTable 3. The Os-DSG-Alc is stable in a lyophilized form and when frozenin solution at−20° C. (40 and 80 μM).

TABLE 3 Osmium Mediator Labels Mediator Concentration Inhibition withLabel Response Antibody Reversal Comments Os-SMCC- Linear PAB IS (≦92%)Yes with BSA-Alc % = Inhibition values ranged Alc PAB DE (≦97%)polyhaptan from 16% to 97% depending on MAB (≦50%) age of Os-SMCC stocksolution. Degrades in solution. Os-SATA- Linear PAB IS (≦44%) Yes withBSA-Alc Stability similar to SMCC. Alc polyhaptan Os-DSG-Alc Linear PABIS (≦91%) Yes with BSA-Alc More stable than conjugate PAB DE ( <87%)polyhaptan made with SATA and SMCC MAB (<78%) crosslinker but stilldegrades in solution. Os-SATA-A₀ Linear Yes with Sheep No with A_(o)HB-A_(o) conjugate was found to be B<HbA_(o)> peptide #1 unstable insolution. Conjugate (≦84%). No was not lyophilized. with Zymed rabbitantibodies

Polyclonal DE (ion-exchange) purified sheep antibody is used in theTinaQuant HbAlc assay. IS (immunosorbent) antibody is prepared usingstandard IS purification methodology. Samples of a monoclonal antibodywere also obtained for evaluation. Inhibition curves were performed insolution with all mixing occurring in microcentrifuge tubes. Assays weremeasured by applying 20 μL onto 6 mm² palladium electrodes with theconditions shown in Table 3. Inhibition curves with the three hemoglobinAlc antibodies (PAB IS, PAB DE, and MAB) were generated by fixingOS-DSG-Alc at 5μM and varying the antibody concentration. Both PAB ISand MAB showed the expected stoichiometric relationship for inhibitionwith the osmium peptide conjugate indicating efficient and fast bindingof the antibody to the Alc peptide. The polyclonal IS and monoclonalboth showed steep inhibition curves with maximum change being reachedclose to 5 μM. Additional antibody above 5 μM showed little effect onincreasing the inhibition. The less purified PAB DE antibody had a muchsmaller slope and as expected required more than 3 times the amount toget close to maximum inhibition. FIG. 6 shows the inhibition curves foreach of the HbAlc antibodies tested. From the inhibition curves we wereable to select reasonable concentrations of antibody for maximumreversal with Alc samples.

Inhibition curves were also performed for the Os-SMCC-Alc (Max=97%) andOS-SATA-Alc (Max=44%) mediator labels. Stability of the mediator labelswere also evaluated by monitoring % inhibition values over time. All ofthe mediator labels showed some degradation when stored in dilutesolutions (40 μM) at RT. Samples frozen at−20° C. appear to be stable.

For demonstrating inhibition reversal, antibody concentrations of 4 μMfor both PAB IS and MAB and 15 μM for PAB DE were chosen from theinhibition curves shown above. Reversal curves were then generated usinga series of dilutions ofBAS-Alc-polyhaptan with a˜1:1 Alc:BSA. TheBSA-Alc acts as our sample and binds to the antibody. FIG. 10 shows thereversal curves for the three antibodies.

While these feasibility studies for a HbAlc immunoassay used an enzymemediated amplification method (Glucdor/PQQ/glucose was used toregenerate reduced mediator after oxidation at the electrode surfaceproviding a higher diffusion controlled current is given by the cottrellequation), they are considered to be indicative of results attainablewith the use of IDA electrodes with bipotentiostatic control is mostpreferred for measuring mediator labeled conjugates in accordance withthis invention.

What is claimed is:
 1. A method for measuring the concentration of oneor more analytes in a liquid sample, said method comprising contacting avolume of said liquid sample with 1) predetermined amounts of at least afirst and second redox reversible species, each respective specieshaving a redox potential differing by at least 50 millivolts from thatof each other species, at least one species comprising a liquid samplediffusible conjugate of a ligand analog of an analyte in the liquidsample and a redox reversible label, said conjugate capable ofcompetitive binding with a specific binding partner for said analyte,and 2) a predetermined amount of at least one specific binding partnerfor each analyte to be measured; and electrochemically determining theconcentration of each of said diffusible redox-reversible species in theliquid sample by contacting said sample with an electrode structureincluding a reference electrode and at least first and second workingelectrodes dimensioned to allow diffusional recycling of the diffusibleredox reversible species in the sample when a predeterminedredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to a second working electrode, saiddiffusional recycling of said species being sufficient to sustain ameasurable current through said sample, applying a first cathodicpotential to the first working electrode and a first anodic potential tothe second working electrode, said first cathodic and anodic potentialscorresponding to those respective potentials necessary to establishcurrent flow through the sample due to diffusional recycling of thefirst redox reversible species without significant interference fromsaid second redox reversible species, measuring current flow at saidfirst anodic and cathodic potentials, applying a second cathodicpotential to said first or second working electrode and a second anodicpotential to the other working electrode, said second cathodic andanodic potential corresponding to those respective potentials necessaryto establish current flow through the sample due to diffusionalrecycling of the second redox-reversible-species without significantinterference from the first redox reversible species, measuring currentflow at said second anodic and cathodic potentials, and correlating therespective measured current flows to that for known concentrations ofthe respective diffusible redox reversible species.
 2. The method ofclaim 1 wherein the cathodic and anodic potentials are applied to theworking electrodes using a bipotentiostat.
 3. The method of claim 1wherein the redox reversible label is a metal ion complex selected fromferrocene and nitrogen-coordinated complexes of transition metal ions.4. The method of claim 1 wherein the redox reversible label is a redoxreversible organic group.
 5. The method of claim 1 for measuring theconcentration of two analytes in a liquid sample wherein the respectiveredox potentials of the first and second redox-reversible-species differby at least 100 millivolts.
 6. The method of claim 1 for measuring theconcentration of one or more analytes in a liquid sample wherein currentflow is measured as at least one of the anodic or cathodic potentials isheld at the predetermined value and the potential of the other is sweptthrough its predetermined value.
 7. The method of claim 1 for measuringtwo proteinaceous analytes in a liquid sample wherein the ligand analogcomponent of the first redox-reversible-species is a peptide comprisingan epitope of a first analyte and the ligand analog component of asecond redox-reversible-species is a peptide comprising an epitope of asecond analyte.
 8. The method of claim 7 wherein one specific bindingpartner is an antibody recognizing the epitope of the first analyte andthe other specific binding partner is an antibody recognizing theepitope of the second analyte.
 9. The method of claim 1 for measuringone analyte in a liquid sample wherein the respective ligand analogcomponent of the first and second redox-reversible-species are differentligand analogs of a single analyte.
 10. The method of claim 9 whereinthe ligand analog component of the first redox reversible species is apeptide comprising a first epitope of the analyte, and the ligand analogcomponent of the second redox-reversible-species is a peptide comprisinga second epitope of the analyte, and the specific binding partners arefirst and second antibodies each recognizing the respective first andsecond epitopes.
 11. A device for detecting or quantifying one or moreanalytes in a liquid sample, said device comprising a sample chamber forholding the liquid sample, at least two redox reversible species locatedfor contact with the liquid sample in the chamber, each redox reversiblespecies capable of diffusion in said liquid sample at least in thepresence of a respective predetermined analyte, said redox reversiblespecies having respective redox potentials differing by at least 50millivolts, and at least one of said redox reversible species comprisinga ligand capable of binding to a specific binding partner for theanalyte, an electrode structure for contact with the liquid sample, saidelectrode structure including a reference electrode and workingelectrodes dimensioned to allow diffusional recycling of a diffusibleredox reversible species in the liquid sample in contact with theelectrode structure when a predeterminedredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to a second working electrode, saiddiffusional recycling of said species being sufficient to sustain ameasurable current through each working electrode, and conductorscommunicating with the respective electrodes for applying said anodicpotential and said cathodic potential and for carrying the currentconducted by the electrode.
 12. The device of claim 11 wherein saidchamber has a sample receiving port and is dimensioned so that it fillsby capillary flow when the liquid sample is contacted with the samplereceiving port.
 13. The device of claim 12 wherein the redox reversiblespecies are located for contact with the liquid sample as it flows intothe chamber.
 14. The device of claim 11 wherein the electrode structurecomprises microarray electrodes selected from the group consisting ofarrays of microdiscs, microbands or microholes.
 15. The device of claim11 wherein the electrode structure comprises interdigitated microarrayelectrodes.
 16. The device of any of claims 11 wherein at least oneredox reversible species includes an osmium complex.
 17. The device ofclaim 11 wherein at least one of the redox reversible species comprisesferrocene or a redox reversible derivative thereof.
 18. The device ofclaim 11 wherein two redox reversible species are positioned for contactwith the liquid sample as it is delivered to the chamber and eachspecies is an osmium complex.
 19. The device of claim 11 including atleast one redox reversible species comprising ferrocene or a redoxreversible derivative thereof and at least one redox reversible speciescomprising an osmium complex.
 20. The device of claim 11 wherein atleast one of the redox-reversible species is an electrochemicallydetectable compound of the formula

wherein R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted, -2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1, 10-phenanthrolinyl, or5,6-disubstituted-1, 10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group, R and R₁ are coordinated to Os throughtheir nitrogen atoms, q is 1 or 0; R₇ is B—(L)_(k)—Q(CH₂)_(i)—; R₂ ishydrogen, methyl, or ethyl when q is 1, and R₂ isB—(L)_(k)—Q(CH₂)_(i)—when q is 0; wherein in the groupB—(L)_(k)—Q(CH₂)_(i)—Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl orethyl; —L—is a divalent linker; k is 1 or 0; i is 1,2,3,4,5 or6; and Bis a group comprising a ligand capable of binding to a specific bindingpartner; Z is chloro or bromo; m is+1 or+2; X is mono or divalent anion;Y is a monovalent anion; and n is 1 or zero, provided that when X is adivalent anion, n is zero, and when m is 1, n is zero and X is not adivalent anion.
 21. The device of claim 11 wherein at least one of theredox reversible species is an electrochemically detectable compound ofthe formula

wherein R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,1 0-phenanthrolinyl, 4,7-disubstituted-1, 10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group, R₅ is 4-substituted-2,2′-bipyridyl or4,4′-disubstituted-2,2′-bipyridyl wherein the substituent is the groupB—(L)_(k)—Q(CH₂)_(i)—and the 4′-substituent is a methyl, ethyl or phenylgroup; R, R₁ and R₅ are coordinated to Os through their nitrogen atoms;Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl or ethyl; —L—is adivalent linker; k is 1 or 0; i is 1, 2, 3, 4, 5 or 6; B is a groupcomprising a ligand capable of binding to a specific binding partner; dis+2 or+3; X and Y are anions selected from monovalent anions anddivalent anions sulfate, carbonate or sulfite wherein x and y areindependently 0, 1, 2, or 3 so that the net charge of X_(x)Y_(y)is−2or−3.
 22. The device of claim 11 wherein the redox reversible specieshave respective redox potentials differing by at least 100 millivolts.23. The device of claim 11 wherein the redox reversible species haverespective redox potentials differing at least 200 millivolts.
 24. Thedevice of claim 11 wherein the device comprises at least two electrodestructures, each in the form of microarray electrodes dimensioned toenable diffusible recycling of a diffusible redox reversible species.25. The device of claim 11 for quantifying a first analyte and a secondanalyte in a liquid sample, said device comprising two redox reversiblespecies, a first redox reversible species comprising a conjugate of aligand analog of the first analyte and a second redox reversible speciescomprising a conjugate of a ligand analog of the second analyte, each ofsaid analyte analog conjugates being capable of binding competitivelywith its respective analyte to a specific binding partner.
 26. Thedevice of claim 25 further comprising a binding partner specific forboth the first analyte and the redox reversible conjugate of the ligandanalog of the first analyte and a binding partner specific for both thesecond analyte and the redox reversible conjugate of the ligand analogof the second analyte said specific binding partners located for contactwith the liquid sample in the chamber.
 27. The device of claim 11further comprising a bipotentiostat in electrical communication with theconductors for applying a redox-reversible-species-dependent-cathodicpotential to one working electrode and aredox-reversible-species-dependent-anodic potential to a second workingelectrode.
 28. The device of claim 27 for quantifying one or moreanalytes in a liquid sample, said device including first and secondredox reversible species, wherein the bipotentiostat is programmable,and it is programmed to apply a first cathodic potential to a firstworking electrode and a first anodic potential to a second workingelectrode, said first anodic and cathodic potentials corresponding tothose potentials necessary to establish current flow through the sampledue to diffusional recycling of the first redox reversible species, andwherein the bipotentiostat is programmed to apply a second cathodicpotential to said first working electrode and a second anodic potentialto the second working electrode, said second cathodic and anodicpotentials corresponding to those potentials necessary to establishcurrent flow through the sample due to diffusional recycling of thesecond redox reversible species, and means for measuring current flowthrough the sample at each of the first and second potentials.
 29. Thedevice of claim 27 for quantifying one or more analytes in a ligandsample, said device including first and second redox reversible species,and at least first and second electrode structures for contact with theliquid sample in the chamber, each of said electrode structurescomprising a microarray of working electrodes, and a switch for changingthe electrical communication of the bipotentiostat between the first andsecond electrode structures.
 30. The device of claim 29 wherein thebipotentiostat is programmable, and it is programmed to apply a firstcathodic potential to a working electrode of the first electrodestructure and a first anodic potential to a second working electrode ofthe first electrode structure, said first anodic and cathodic potentialscorresponding to those necessary to establish current flow through thesample due to diffusional recycling of the first redox reversiblespecies, and wherein the bipotentiostat is programmed to apply a secondcathodic potential to a working electrode of the second electrodestructure and a second anodic potential to a second electrode of thesecond electrode structure, said second cathodic and anodic potentialcorresponding to those potentials necessary to establish current flowthrough the sample due to diffusional recycling of the second redoxreversible species, and means for measuring current flow through thesample at each electrode structure.
 31. The device of claim 11 whereinthe first and second reversible species each comprise a conjugate ofdifferent ligand analogs of one analyte, each of said conjugates capableof binding competitively with said analyte to one of two independentspecific binding partners for said analyte.
 32. The device of claim 11for quantifying glycosylated hemoglobin wherein at least one of the tworedox reversible species comprises a conjugate of the formulaGluc-Val-His-Leu-Thr - L - M₁ wherein M₁ is a redox reversible label, Lis a linker and Gluc-Val-His-Leu-Thr- is the N-terminal sequence of theβ-chain of hemoglobin Al c.
 33. The device of claim 32 wherein the redoxreversible label is a metal ion complex.
 34. The device of claim 32wherein M₁ is an osmium ion complex or ferrocene.
 35. The device ofclaim 32 wherein the other reversible redox species comprises a redoxreversible conjugate of the formula Val-His-Leu-Thr - L -M₂ wherein M₂is a redox reversible label and L is a linker.
 36. The device of claim35 wherein the redox reversible label is a metal ion complex.
 37. Thedevice of claim 35 wherein the redox potential of M₁ and M₂ differ by atleast 100 millivolts.
 38. The device of claim 35 wherein the redoxpotential of M₁ and M₂ differ at least 200 millivolts.
 39. The device ofclaim 35 further comprising a specific binding partner for bothhemoglobin Alc and the redox reversible conjugate Gluc-Val-His-Leu-Thr-L-M₁, said specific binding partner located for contact with the samplein the chamber.
 40. The device of claim 39 further comprising a specificbinding partner for both hemoglobin and the redox reversible conjugateVal-His-Leu-Thr -L M₂, said specific binding partner located for contactwith the sample in the chamber.
 41. A kit for measuring theconcentration of one or more analytes in a liquid sample, said kitcomprising at least two redox reversible species for contact with theliquid sample, each capable of diffusion in the liquid sample at leastin the presence of a predetermined analyte, at least one speciescomprising a conjugate of a ligand analog of an analyte and a redoxreversible label, said redox reversible species having respective redoxpotentials differing by at least 50 millivolts; a specific bindingpartner for each analyte; an electrode structure for contact with theliquid sample, said electrode structure including a reference electrodeand working electrodes dimensioned to allow diffusional recycling ofdiffusible redox reversible species in the sample when a predeterminedredox-reversible-species-dependent-cathodic potential is applied to oneworking electrode and a predeterminedredox-reversible-species-dependent-anodic potential is applied to thesecond working electrode, said diffusional recycling of said speciesmeans sufficient to sustain a measurable current through the sample; andconductors communicating with the respective electrodes for applyingsaid anodic potential and said cathodic potential and for carrying thecurrent conducted by the electrodes.
 42. The kit of claim 41 wherein theelectrode structure comprises microarray electrodes selected from thegroup consisting of arrays of microdiscs, microbands, or microholes. 43.The kit of claim 41 wherein the electrode structure comprisesinterdigitated microarray electrodes.
 44. The kit of claim 41 whereinthe redox reversible species are mixed as a composition for contact withthe liquid sample.
 45. The kit of claim 41 wherein the redox reversiblelabel of at least one redox reversible species comprises an osmiumcomplex.
 46. The kit of claim 41 wherein the redox reversible specieshave respective redox potentials differing by at least 100 millivolts.47. The kit of claim 41 wherein the redox reversible species haverespective redox potentials differing by at least 200 millivolts.